The exchange of macromolecules and nutrients across mammalian microvessel endothelial cells is mediated in part by a system of cytoplasmic and membrane bound vesicles. This mode of blood-tissue exchange may play a significant role in maintaining normal tissue function. Yet the mechanism, regulation and specificity of the vesicular mode of capillary permeability is poorly understood due to the difficulty in studying this process. We have developed new methodologies which permit the accurate quantitation of endocytosis and exocytosis in vitro using both freshly isolated microvessel endothelial cells and cultured endothelium. We have developed techniques for fluorescently labelling numerous solutes to biochemically analyze the two major steps in vesicular transport, endocytosis and exocytosis. The purpose of this research project is to determine the basic mechanisms which exist for the regulation and specificity of vesicle-mediated transendothelial solute transport. Using microvessel endothelial cells isolated from rat epididymal fat we will examine the regulation of vesicle mediated transport by serum proteins, hormones, growth factors and pathologic conditions such as hypoxia and hyperoxia. These experiments will examine whether changes in vesicle-mediated transport play a role in the observed microvascular leakiness during conditions such as inflammation, wound healing and diabetes. We will examine the specificity of vesicle-mediated transport. Experiments are designed to determine whether microvessel endothelial cells utilize specific hormone receptors to facilitate the transendothelial transport of selectively adsorbed solutes. The specificity of vesicle mediated transport for molecules of different molecular weight, shape, charge and carbohydrate content will be evaluated. Results of these experiments will be related to the observed changes in vascular permeability observed in diabetes mellitus and atherosclerosis.